Competition of electrostatic and hydrophobic interactions in the native state of

To further probe the strength of distant N- and C-terminal electrostatic interactions in the A30P and A53T mutants upon destabilization of the long-range hydrophobic cluster, we sought to determine the affinities of these proteins for the polycation spermine. The rationale

was that if electrostatic contacts are modulated, one should perceive a change in the affinity of the protein for the polycations.

When we titrated αS A30P and A53T mutants with spermine we observed a general effect of the polycation on the amide resonances of the protein. Both the chemical shift perturbations and the changes in the intensity of the signals observed for wt αS (Fernandez et al., 2004) were reproduced for the A30P and A53T mutants (Figure 5.12). The chemical shifts perturbations were also localized at the C-terminus of the protein, with the principal changes centered on the stretch comprising residues 124 to 135, similar to the wt protein, and thus permitting a comparison of the binding affinities for the three proteins.

Figure 5.12. Binding of spermine to mutant αS is similar as to the wt protein. Comparison between polyamine binding to wt and mutant αS. Upon spermine binding (3mM) to αS, changes in ¹⁵N chemical shifts (bold black line) and in peak intensities (colored bars) are visible, reflecting formation of a complex between the negatively charged C-terminus of the protein and the polycation. A. αS-wt. B. αS-A30P. C. αS-A53T.

If the protein is in fast exchange with the ligand, due to the conformational average sampled by the timescale of the NMR measurement, the chemical shifts of those residues directly implicated in the binding event should gradually change from 0 until a maximum

upon saturation. Thus one may employ the HSQC-based titration experiments to evaluate the dissociation constants (Kd) of the formation of the protein-ligand complex.

The determinations of the Kd for the A30P and the A53T proteins is shown in figure 5.13. A global analysis, assuming a single Kd but independent maximal chemical shifts perturbations for each residue evaluated, determined that both mutant variants have ~ half the affinity for spermine than the wt protein (1.3 mM and 1.23 mM for A30P and A53T, respectively, vs. 0.61 mM for αS wt).

Figure 5.13. Determination of dissociation constants for spermine-mutant αS complexes. Kd for the binding of the polycation spermine (+4) to both A30P and A53T αS proteins were determined by titration experiments based on ¹H-¹⁵N-HSQC NMR spectra. A. ¹⁵N chemical shifts difference as a function of total concentration of ligand for αS-A30P (left) and αS-A53T (right). Residues are as follows: Ala¹²⁴ (dark red), Glu¹²⁶ (orange), Met¹²⁷ (light green), Ser¹²⁹ (dark green), Asp¹³⁵ (cyan). B.

Comparison of Kds for spermine-αS complexes determined by this procedure. The values are 0.61 ± 0.03 mM for αS-wt, 1.31 ± 0.05 mM for αS-A30P, and 1.23 ± 0.06 mM for αS-A53T. Protein concentration was 100 μM in all the cases.

This result would suggest that the N-terminal and the polycation compete for the negatively charged C-terminal, and that the composite influence of the electrostatic interactions reduce the affinity of the protein for the polycation. The net charge of the terminal domain is + 4, equivalent to a single molecule of spermine. The 100 μM of N-terminus in solution was considerable lower than the high excess of polycation present in the sample in great excess. Thus if competition were the sole cause of the affinity change, the Kd of the N-terminus/C-terminus complex would have to be ~ 10 times lower than for the polyamine/C-terminus complex. This could be explained in terms of the high local concentration effect that the charges at the N-terminus are expected to exert since they are directly attached to the C-terminus through the polypeptide chain and are not subject to diffusion or exchange as the polyamine molecules do.

A B

In the previous we must consider that there is a second effect that may additionally regulate the affinity of the C-terminus to the polycation, namely the residual structure of the C-terminus, which is indeed perturbed in the mutants, as revealed by the RDCs.

In order to explore the possibility that residual structure at the C-terminus regulates binding to polyamines, we studied the spermine binding characteristics of a peptide comprising residues 105 to 136 derived from the C-terminus of αS. As probed by RDCs this peptide populates unfolded conformations with diminished residual structure when compared with the full length protein (Chapter 1), and thus is an excellent probe for evaluating polyamine binding in the absence of both long range interactions.

Upon titration with spermine the peptide displayed essentially a similar dose-dependent shift of the resonances, although the saturating chemical shifts perturbations for the evaluated residues were 50% to 80% higher than the values displayed by the full length protein. The stronger chemical shift perturbation in the peptide could be due to the absence of conformational restrictions imposed by the full length polypeptide chain.

However, the resulting Kd for the peptide/spermine complex was ~ 1 mM, approximately 80% higher than the 0.6 mM determined for the wt protein. These findings support the notion that the lost of residual structure at the C-terminus is able to modulate binding to ligands, which may also have consequences for other protein-protein interactions.

Figure 5.11. Determination of dissociation constants for spermine- αS terminal peptide complex. A titration experiment for spermine binding to the C-terminal peptide of αS (αS105-136) was carried out to determine the Kd for the formation of the complex. ¹H-¹⁵N-HSQC NMR spectra were acquired at a constant concentration of peptide (1 mM) and different concentration of polycation. A. ¹⁵N chemical shifts difference as a function of total concentration of ligand for αS-C-terminal peptide.

Residues are as follows: Ala¹²⁴ (dark red), Glu¹²⁶ (orange), Met¹²⁷ (light green), Ser¹²⁹ (dark green), Asp¹³⁵ (cyan). B. Comparison of Kds for complexes of spermine-αS (0.61

± 0.03 mM) and for spermine-αS-C-terminal peptide (1.0 ± 0.2 mM).

A B

If we now compare the whole picture, the doubling in Kd of the mutant protein/spermine complex can be attributed to the loss of residual structure and from the strengthen of electrostatic long range interactions. Assuming a linear combination of both effects we can estimate that the competition between the N-terminus and the polyamine for the negative charges at the C-terminus would account for ~ 20 % increase in the Kd. Thus there is an increment in the effectiveness of the N-terminal/C-terminal interaction upon reduction of the long range hydrophobic interaction.

Figure 5.12. Synergistic long range interactions in αS. Proposed scheme for the occurrence of long-range interactions in αS. The ensemble of conformations populated by native αS (center) accounts for long-range interactions involving the N-terminus (blue), the NAC region (yellow) and the C-N-terminus (red) of the protein. The presence of genetic mutations (right) abolishes the hydrophobic NAC/C-terminus interaction, while charge shielding (right) as the presence of salt or low pH cause reduction of the electrostatic N-/C-terminus interaction.

In conclusion, dipolar couplings demonstrate that the intricate network of long-range interactions is perturbed in PD-linked mutant αS, such that the protein is more flexible and able to sample conformations somewhat more collapsed that the wt protein. Due to the preferential interaction with the N-terminus, the C-terminus would not be favored to interact with the NAC region, and the hydrophobic residues of this latter domain would be exposed to the solvent, increasing the free energy of the ensemble of conformations. Thus the A30P and A53T mutant αS may overcome more easily the energetic barrier for self-association, leading to an increased tendency to oligomerize (Conway et al., 2000).

6. Results. Chapter III: “Structural characterization of the PD-associated protein